Sulfhydryl oxidase (SOX) is an enzyme known to catalyze the conversion of thio compounds to the corresponding disulfides according to the equation: 2R-SH+O.sub.2 .fwdarw.R-S-S-R+H.sub.2 O.sub.2. SOX is therefore of interest in applications where oxidation of free sulfhydryl groups to disulfide linkages is sought.
Non-specific oxidants such as hydrogen peroxide, peracids, borates, bromides, etc. which have been employed heretofore for effecting disulfide bond formation are disadvantagous as unwanted side reactions may occur. In contrast enzyme catalyzed reactions, such as the oxidation of free sulfhydryls by the enzyme SOX, can provide the selectivity desired avoiding side reactions. For example the flavour problems caused by non-specific oxidants due to oxidation of other components of a food system can be eliminated due to the specificity of an enzyme catalyzed reaction. Further advantages of SOX are that SOX oxidizes under milder conditions than non-specific oxidants, which is useful in food systems, and that the use of highly acidic, highly basic or high temperature conditions in food systems is not required for SOX activity. In contrast such conditions are necessary when non specific oxidants are used and such conditions may have an adverse effect on the organoleptic quality of the food system. Furthermore an enzyme catalyzed oxidation will also usually have a greater velocity than an oxidation catalyzed by a non-specific oxidant.
One example of a process in which treatment with sulfhydryl oxidase is of value is the removal of a burnt flavour from Ultra-High Temperature (UHT) sterilized milk. For details of such usage of SOX, reference is made to U.S. Pat. Nos. 4,087,328 and 4,053,644.
Another example of a process in which treatment with sulfhydryl oxidase is beneficial is a process in which SOX is employed as a dough conditioner to act on free sulfhydride groups in contrast to non-specific oxidants. In U.S. Pat. No. 4,894,340 it is suggested that Aspergillus niger SOX could improve the rheological properties of dough and provide an improvement in the form and texture of a baked product.
SOX derived from a mammalian (bovine) source can be used in a process for removal of burnt flavour from milk but does not exhibit beneficial effects in a wheat flour dough [Kaufman et al. Cereal Chem. 64 (3): 172-176]. Microbially derived SOX can be used in both processes.
Several enzymes derived from both mammalian and microbial sources having the ability to catalyze the conversion of thio compounds to the corresponding disulfides according to the equation: 2R-SH+O.sub.2 .fwdarw.R-S-S-R+H.sub.2 O.sub.2 have been reported in the scientific literature.
In 1975 Janolino and Swaisgood purified an iron-dependent sulfhydryl oxidase from bovine milk, said oxidase demonstrated activity toward GSH, cysteine, dithiotreitol, 2-mercaptoethanol and reduced ribonuclease A. Milk extracts from other sources, including a human source [Isaacs C. E. et al., Pediatr. Res. 18:532 (1984)] have also been reported to exhibit sulfhydryl oxidase activity.
Further sources of sulfhydryl oxidase that have been reported are kidney homogenates and mammalian pancreas tissue [Clare D. A. et al., Arch. Biochem. Biophys. 230:138 (1984)].
Sulfhydryl oxidase activity has also been discovered in rat epididymal fluid [Chang T. S. K. and Morton B., Biochem. Biophys. Res. Commun. 66:309 (1975)]. The best substrates for this sulfhydryl oxidase were reported to be dithiotreitol, GSH and cysteine. As in the case of the skin and bovine milk enzymes, this rat enzyme was capable of reactivating reduced ribonuclease A.
Mammalian source SOX e.g. as mentioned in the U.S. Pat. Nos. 4,087,328 and 4,053,644 has the disadvantage that it is not available in large quantities at economic prices and furthermore cannot be used in a process for improving a dough.
Therefore research has been directed at a microbial source that could possibly provide a readily available commercially attractive supply of SOX. Various microbial sources of sulfhydryl oxidase-are known.
In 1956 Mandels G. R., J. Bacteriol, 52:230 (1956) reported that the spores of the fungus Mirothecium varrucaria contained a sulfhydryl oxidase which catalyzed the oxidation of reduced glutathione (GSH), cysteine and homocysteine with concomitant reduction of H.sub.2 O.sub.2.
In 1975 Olson J. A. [Ph.D. dissertation, University of Iowa (1976)] isolated a sulfhydryl oxidase from the culture fluid of an organism believed to be Dactylium dendroides. This copper metallo enzyme was found in mycelium extracts which also contained galactose oxidase. Olson reported that the purified sulfhydryl oxidase was capable of reactivating reductively denatured galactose oxidase.
Microbial SOX can be isolated from commercial enzyme preparations comprising SOX as a side product or contaminant obtained from microorganisms known to produce SOX. Examples of such commercial enzyme preparations are Fungamyl.sup.R, Pectinex.sup.R, and some amyloglucosidases However, separation costs and yield loss in purification and/or concentration of the SOX make the SOX product from such sources prohibitively expensive.
It is clear that separation costs and yield loss in purification and/or concentration of SOX recovered from the culture medium of a microorganism or from the microorganism itself will be uneconomical when such a microorganism is only capable of moderate levels of SOX production.
In U.S. Pat. No. 4,632,905 of Starnes et al. it is claimed that the microbial species Aspergillus sojae, Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, and Penicillium lilacinum produce SOX at levels high enough for potential recovery of SOX. Starnes at al. also reported that very low levels of SOX elaboration were detected for Bacillus licheniformis, Bacillus coagulans, Bacillus acidopullulyticus, Bacillus stearothermophilus, Mucor miehei, and Trichoderma reesei. Subsequently they described that in particular it was possible to recover high unit activity SOX products through cultivation of Aspergillus sojae.
They describe that the oxidase was elaborated both intracellularly and extracellularly in recoverable quantities when the microorganism was cultivated. The cells could be easily removed from the whole broth (by conventional methods, e.g., centrifugation) and the cell-free broth could be filtered and usually concentrated by diafiltration with an overall recovery of about 40%. The SOX enzyme could also be recovered from the cells in similar overall yields by the same recovery protocol following rupturing of the microbial cell by high pressure disruption, sonication, enzymatic digestion or simply by cell autolysis. In general the same methods heretofore employed to liberate and produce solutions of other intracellular fungal enzymes from various Aspergillus species were used.
In U.S. Pat. No. 4,894,340 of Hammer et al., 1990, an isolated sulfhydryl oxidase enzyme derived from Aspergillus niger is described. This microbial SOX is characterized by a pH-optimum of about 5.5. The method for recovering said Aspergillus niger SOX is similar to the method described in the cited Starnes patent and comprises cultivating a SOX producing strain of the fungus, recovering SOX from the fungus, and purifying the recovered SOX. The recovery of SOX can be accomplished by lysing the cells by enzymatic digestion or other suitable means and precipitating the resulting proteins, or by suspending A. niger in brine of sufficient strength to partition the enzyme into the brine solution.
The present methods of obtaining sulfhydryl oxidase of mammalian origin are too lengthy, complex and expensive for economically feasible production of the enzyme.
From the literature the only apparent economically feasible sources of microbial sulfhydryl oxidase are Aspergillus sojae and Aspergillus niger. In practice however no commercial preparations of pure sulfhydryl oxidase are available. The production and purification of the product are still too complex and costly.
The subject invention is aimed at solving the abovementioned problems and is directed at the production of mammalian or microbial forms of sulfhydryl oxidase in a process that is economical and can lead to easier production of pure forms of the desired enzyme. Furthermore, with the process according to the invention it is possible to produce large amounts of many different forms of sulfhydryl oxidases that could not be produced previously. It is also possible to obtain sulfhydryl oxidases from Aspergillus niger and Aspergillus sojae in larger amounts and requiring less purification than has been described in the cited U.S. Patents.